1. Field of the Invention
The present invention relates to novel amino acid fluorides and protected amino acid fluorides and their use in synthetic biochemistry, including peptide syntheses. More particularly, this invention is directed to the N-protected amino acid fluorides, and free amino acid fluorides and the hydrogen fluoride salts thereof, the side chain of which may be unprotected or protected with a blocking group and their use thereof in peptide synthesis.
2. Background of the Prior Art
As more and more polypeptides become of medicinal importance, there is an increasing incentive to improve the methods by which they may be synthesized. In recent years, peptides which have been found to be of possible pharmacological importance include those active against various diseases, such as cancers, diabetes, and plant toxins, etc. Others have shown specific activity as growth promoters or suppressants, antibiotics, insecticides, contraceptives, anti-hypertensives, sleep-inducers, anti-depressants, analgesics, etc. The list is long and varied.
Currently, syntheses of peptides in solution by classic or various repetitive methods or on a solid support (Merrifield) are popular techniques. Solution methods have the advantages of being easily monitored and allowing purification of intermediates, if necessary, at any stage. A major drawback is the relative slow pace of the synthesis with each step being carried out manually.
The major advantage of the Merrifield Method is its easy automation so that unattended, computer-controlled machine synthesis is possible. Unfortunately, this method suffers from an inherent deficiency due to the insoluble nature of the support on which the synthesis proceeds. Unless each acylation step occurs with 100% efficiency, mixtures will inevitably be built up on the polymer. The longer the chain, the greater will be the contamination due to undesired side reactions. Products produced in such reactions remain to contaminate the desired product when at the end of the cycle it is removed from the polymeric matrix. The properties of these peptides will not differ sufficiently for peptides of greater than about 30-40 residues to make efficient separation feasible.
For very long segments (50 or more amino acids), therefore, current methods are not satisfactory. Often mixtures are obtained of such forbidding complexity that it may be difficult or impossible to isolate the desired peptide.
The problems enumerated hereinabove could be eliminated if the proper derivatives of the underlying amino acids and the proper reaction conditions could be found.
For example, FMOC, (Nxcex1-(9-fluorenylmethyl)-oxycarbonyl), protected amino acid chlorides, which are described by Carpino, et al. in J. Org. Chem. 51, 3732 (1986) have been used as acylating agents for stepwise peptide syntheses for both solution and solid phase techniques.
However, the amino acid chlorides have major drawbacks associated therewith. First, the acid chlorides react with trace amounts of water, such as moisture in the air, to give the corresponding amino acid. Therefore, they are not so stable, and as such, they are not a prime candidate for long term storage. Consequently, an objective was to find an amino acid derivative which was stable to moisture.
Moreover, another problem associated with amino acid chlorides is that it has not been possible to date to synthesize amino acid chlorides in which the protecting groups on the side chains of the amino acids can be removed under extremely mild conditions. As one skilled in the art is well aware, many of the amino acids have functional groups on the side chains which can interfere with peptide formation unless otherwise protected. In peptide synthesis, only the mildest conditions should be used to remove these protecting groups. For example, one of the easiest protecting groups to remove from the side chains containing amino, hydroxyl or carboxyl functions, such as lysine, tyrosine, threonine, serine, aspartic acid, glutamic acid and the like, is t-butyl or t-butyl containing moieties. For example, trifluoracetic acid can easily remove the t-butyl group from a serine side chain; on the other hand, a benzyl protecting group on the side chain can not be removed by said treatment but instead requires a more potent acid such as HF or trifluoromethanesulfonic acid. Therefore, the conditions for removing the benzyl group from the side chain are much harsher relative to the t-butyl groups. Furthermore, the mild catalytic hydrogenolysis of benzyl groups is not generally applicable to long chain peptides or resin attached peptides.
Although benzyl groups on the side chains of N-protected amino acid chlorides, can be prepared, such as FMOC-cysteine-S-benzyl chloride, FMOC-lysine-xcex5 carbobenzoxy chloride, FMOC-tyrosine-O-benzyl chloride, FMOC-serine-O-benzyl chloride and FMOC aspartic acid xcex2-benzyl ester, these molecules suffer from the disadvantages described hereinabove. Consequently, an investigation was commenced to determine if t-butyl or xe2x80x9ct-butyl likexe2x80x9d containing groups can be used to protect the side chain of amino acid chlorides. Unfortunately, efforts in this area were unsuccessful. None of the above compounds could be synthesized if the t-butyl group was used in place of the benzyl substitution.
This was not unusual since it is well known that t-butyl-based protecting groups are readily deblocked by hydrogen chloride which is an inevitable by-product of acid chloride formation and/or long term storage (hydrolysis by trace amounts of water). For example, in the case of the FMOC-tyrosine derivative 1, the acid chloride could be obtained, but after several days it was noted to lose the t-butyl group slowly. 
Furthermore, compound 1 as well as the analogous serine and threonine derivatives could be obtained only as oils which could not be crystallized and were therefore difficult, if not impossible, to purify.
In the case of the FMOC aspartic acid derivative 4, treatment with thionyl chloride gave only the aspartic acid anhydride 6, presumably via the unstable acid chloride 5 which undergoes intramolecular loss of t-butyl chloride. 
Lysine derivative 7 could not be converted to an acid chloride because of the marked sensitivity of the BOC function. 
Similar problems arise in the cases of Arg, His, Asn, Gln, and Trp. The net result of these problems is that only about one half of the commonly occurring amino acids can be converted to stable amino acid chlorides.
Therefore, a search was undertaken to find an amino acid candidate for use in peptide synthesis which is inexpensive, stable to moisture, and which shows great potential for long-term storage. Moreover, it was hoped that a candidate could be found where the protecting groups on the amino acid side chain could be removed under milder conditions then those used to remove the benzyl group. Preferably, it was hoped that a t-butyl containing group or a group as easily removable as t-butyl could be placed on the side chain of these amino acid candidates.
The present invention circumvents the difficulties experienced with respect to the acid chlorides and accomplishes the goals described hereinabove. The compounds of the present invention are effective in coupling with amino acids or peptides to form new peptide bonds. Moreover, the compounds of the present invention are more stable to moisture then the acid chlorides and therefore can be used for long term storage. Furthermore, t-butyl containing protecting groups and other protecting groups can be placed on the side chains of these amino acid compounds and removed under milder conditions than those required for the removal of benzyl groups. Finally, the compounds of the present invention are potent acylating agents in peptide bond formation.
These compounds are, much to our surprise, the corresponding amino acid fluorides.
The present invention is directed to an amino acid of the formula: 
or the hydrogen fluoride salts thereof wherein
BLK is an N-amino protecting group;
AA is an amino acid residue; and
X isxe2x80x94or a protecting group.
The present invention is also directed to a method for preparing a peptide which comprises reacting the amino acid fluoride described hereinabove with an amino acid or peptide having a free amino group and removing the protecting groups therefrom.
In addition, the present invention is directed to a method for preparing amino acid fluorides using a new fluorinating agent, a fluoroformamidinium salt, of the formula: 
wherein R15, R16, R17 and R18 are independently lower alkyl, aryl, aryl lower alkyl, cycloalkyl, cycloalkyl lower alkyl or R15 and R16 taken together with the nitrogen atom to which they are attached form a 5- or 6-membered ring or R17 and R18, taken together with the nitrogen atom to which they are attached form a 5- or 6-membered ring or R15, R16, R17 and R18 taken together with the nitrogen atom to which they are attached form a 5- or 6-membered ring and Axe2x88x92 is a counter ion.
The present invention is also directed to use of the fluoroformamidinium agent as a coupling agent for the assembly of peptides.
As used herein, the term xe2x80x9camino acidxe2x80x9d refers to an organic acid containing both a basic amino group (NH2) and an acidic carboxyl group (COOH). Therefore, said molecule is amphoteric and exists in aqueous solution as dipole ions. (See, xe2x80x9cThe Condensed Chemical Dictionaryxe2x80x9d, 10th ed. edited by Gessner G. Hawly, Van Nostrand Reinhold Company, London, Eng. p. 48 (1981)). The preferred amino acids are the xcex1-amino acids. They include but are not limited to the 25 amino acids that have been established as protein constituents. They must contain at least one carboxyl group and one primary or secondary amino group on the amino acid molecule. They include such proteinogenic amino acids as alanine, valine, leucine, isoleucine, norleucine, proline, hydroxyproline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, hydroxylysine, ornithine, arginine, histidine, penicillamine and the like.
An xe2x80x9camino acid residuexe2x80x9d, as defined herein, is an amino acid minus an amine hydrogen on the amino end of the molecule and the OH group on the carboxy end of the molecule (i.e., it includes the acyl group 
on the carboxy end of the molecule). Therefore, unless designated to the contrary, the group xe2x80x9cAAxe2x80x9d signifies an amino acid residue. For example, the amino acid residues of various amino acids are represented below:
Therefore, the symbol xe2x80x9cAA-Fxe2x80x9d refers to an amino acid fluoride, i.e., a compound having a fluoro group attached to the acyl group 
of the amino acid
When BLK is hydrogen, then the structure becomes the amino acid fluoride of an amino acid having a free amino group. However, in view of the synthesis of the amino acid fluoride described hereinbelow, the free amino acid fluoride may be isolated as the hydrogen fluoride salt thereof.
It will be apparent to one skilled in the art, shown by exemplification in the table hereinabove that in the course of protein synthesis, it may be necessary to protect certain side chains of the amino acids to prevent unwanted side reactions. For example, it may be necessary to protect the hydroxyl group on the side chain of tyrosine, serine, or threonine in order to prevent these groups from interfering with the desired reactions. This is a common problem in peptide synthesis and many procedures are available for protecting the functional groups on the side chains of the amino acids. Such procedures for protecting various functional groups are known to one skilled in the art and are described in the treatise entitled xe2x80x9cThe PEPTIDESxe2x80x9d, Vol. 2, Edited by E. Gross and J. Meienhoffer, Academic Press, NY, N.Y., pp. 166-251 (1980), and the book entitled xe2x80x9cReagents for Organic Synthesisxe2x80x9d, by T. W. Green, John Wiley and Sons, New York, 1981, the contents of both being incorporated herein by reference.
For example, when the functional side chain contains an hydroxy group, such as threonine or serine, it can be protected by such groups as methyl, methoxymethyl(MOM), 2-methoxyethoxymethyl(MEM), tetrahydropyranyl, xcex2-trimethylsilylethyl, 4-methoxytetrahydropryanyl, 1-ethoxyethyl, t-butyl, p-methoxybenzyl, p-halobenzyl, o-nitrobenzyl, p-nitrobenzyl, o-chlorobenzyl, adamantyl, diphenylmethyl, triphenylmethyl, cyclohexyl, cyclopentyl, 1-benzyloxycarbonyl, tri-substituted silyl, wherein the substituents are independently aryl, alkyl or aralkyl, 2,2,2-trifluoroethyl, and the like. The preferred groups for the protection of the hydroxyl side chain are adamantyl, t-butyl, 4-methoxybenzyl, cyclopentyl, and cyclohexyl.
When the side chain contains a phenol, such as intyrosine, it may be protected by such groups as methyl, methoxymethyl(MOM), methoxyethoxymethyl(MEM), xcex2-trimethylsilylethyl, methylthiomethyl, tetra-hydropyranyl, isopropyl, cyclohexyl, cyclopentyl, t-butyl, adamantyl, 4-methoxyphenylsilyl, o-nitrobenzyl, 2,4-dinitrophenyl, m-bromobenzyl, 2,6-dichlorobenzyl, trisubstituted-silyl wherein the substituents are independently alkyl, aryl or aralkyl, ethoxycarbonyl, carbamoyl and the like. The most preferred protecting groups are adamantyl, 4-methoxybenzyl, t-butyl, cyclopentyl, and cyclohexyl. An especially preferred protecting group is t-butyl.
A carboxy side chain, such as that found in aspartidc acid or glutamic acid, can be protected by the following groups: 1- or 2-adamantyl, methoxymethyl, methythiomethyl, t-butyl, methyl, ethyl, phenyl, tetrahydropyranyl, cyclopentyl, cyclohexyl, cycloheptyl, 4-picolyl, trisubstituted-silyl wherein the substituents are independently alkyl, aryl or aralykl, N-piperidinyl, N-succinimidoyl, xcex2-trimethylsilylethyl, 4-methoxybenzyl, benzyl, p-bromobenzyl, p-chlorobenzyl, p-nitrobenzyl, phenacyl, N-phthalimidoyl, 4-alkyl-5-oxo-1,3-oxazolidinyl, trisubstituted-stannyl wherein the substituents are independently alkyl, aryl or aralkyl, and the like. The most preferred protecting group is t-butyl.
If the functional group on the side chain is mercapto, e.g., cysteine, such groups as tri-phenylmethyl, benzyl, 4-methylbenzyl, 3,4-dimethylbenzyl, 4-methoxybenzyl, xcex2-trimethylsilylethyl, p-nitrobenzyl, 4-picolyl, diphenylmethyl, triphenylmethyl, bis(4-methoxyphenyl)methyl, diphenyl-4-pyridylmethyl, 2,4-dinitrophenyl, t-butyl, t-butylthio, adamantyl, isobutoxymethyl, benzylthiomethyl, thiazolidinyl, acetamidomethyl, benzamidomethyl, 2-nitro-1-phenylethyl, 2,2-bis(carboethoxy)ethyl, 9-fluorenemethyl, acetyl, benzoyl, and the like can be used to protect said group. In this case, it is preferred that the protecting groups are t-butyl, t-butylthio, 4- methoxybenzyl, and triphenylmethyl.
If the side chain contains an amino group, such as the xcex5-amino group of lysine and ornithine, the following groups may be used: 9-fluorenylmethyloxycarbonyl, 9-(2-sulfo)fluorenylmethyloxycarbonyl, xcex2-trimethylsilyl ethyloxycarbonyl, 2-furanylmethyloxycarbonyl, adamantyloxycarbonyl, carbobenzoxy, t-butyloxycarbonyl, t-amyloxycarbonyl, cyclobutyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, 1-methylcyclohexyloxycarbonyl, isobornyloxycarbonyl, benzyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, chlorobenzyloxycarbonyl, isonicotinyloxycarbonyl, p-toluenesulfonylamidocarbonyl, methylsulfonylethyloxycarbonyl, xcex2,xcex2,xcex2-trichloro-ethyloxycarbonyl, dithiasuccinoyl, phthaloyl, 4,5-diphenyl-4-oxazoline-2-one, piperidino oxycarbonyl trifluoroacetyl, chloroacetyl, p-toluenesulfonyl and the like. Preferred groups for the protection of this amino side chain are carbobenzoxycarbonyl, t-butyloxycarbonyl, and adamantyloxycarbonyl.
If the amino acid has an imidazole group, such as in histidine, the following groups may be used to protect the side chain: benzyloxymethyl, piperdinylcarbonyl, phenacyl, pivaloyloxymethyl, 1-(alkoxycarbonylamino)-2,2,2-trifluoroethyl, 1-trifluoromethyl-1-(p-chlorophenoxy-methoxy)-2,2,2-trifluoroethyl, 2,4-dinitrophenyl, toluenesulfonyl, FMOC, triphenylmethyl, t-butyloxycarbonyl, t-butyloxymethyl and the like. The preferred groups are FMOC, t-butyloxycarbonyl, triphenylmethyl, and t-butyloxymethyl.
When the amino acid has a guanidine side chain, such as in arginine, the following protecting groups can be used to protect the xcfx89-nitrogen on the guanidine moiety: methoxytrimethylbenzenesulfonyl, pentamethylchromane-sulfonyl, mesitylenesulfonyl, tolunesulfonyl, 2,4,6-trimethylbenzenesulfonyl, trimethoxybenzensulfonyl, bisadamantyloxylcarbonyl, nitro, tosyl, and the like. The preferred protecting groups are methoxytri-methylsulfonyl, pentaamethylchromanesulfonyl, bisadamantyloxycarbonyl, and mesitylenesulfonyl.
For side chains containing an amide group such as in glutamine and asparagine, the following groups can be used to protect the side chain: dimethoxybenzyhydryl, 9-xanthenyl, 2,4,6-trimethoxybenzyl, and the like.
As used herein in the instant specification, the term xe2x80x9calkylxe2x80x9d, when used alone or in combination with other groups refers to a carbon chain containing from 1 to 6 carbon atoms. They may be straight chains or branched and include such groups as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, amyl, n-hexyl and the like. The preferred alkyl group contains from 1-3 carbon atoms. The term aryl as used herein refers to an aromatic ring system containing from 6-10 ring carbon atoms and up to a total of 15 carbon atoms. It includes such groups as phenyl, xcex1-naphthyl, xcex2-naphthyl, and the like. The preferred group is phenyl.
Aralkyl groups are aryl groups attached to the main chain through an alkylene bridge. Such groups include benzyl, phenethyl and the like. The preferred aralkyl group is benzyl.
The aryl and aralkyl groups herein may be unsubstituted or may be substituted with an electron donating group in situations wherein the protecting group is cleaved by acid. An electron donating group as defined herein shall be interpreted as a group that will release or donate electrons more than hydrogen would if it occupied the same position in the molecule. See, J. Marsh, Advance Organic Chemistry, 2nd Ed., McGraw Hill, Inc. (1977). These types of groups are well known in the art. Examples of electron donating groups include alkyl, lower alkoxy, aralkoxy; and the like. These electron donating groups, e.g., alkoxy, may be present on the aryl moiety of the following groups: DMB, TMB, Mtr, Pmc, Bz, Trt, CBZ and the like.
The protecting groups described hereinabove are well known to one skilled in the art. They can be removed under very mild acidic or basic conditions. The preferred protecting groups are those which can be cleaved by acid or base under conditions which are milder than those used to cleave the benzyl group. These include groups which can be cleaved by trifluoroacetic acid at room temperature within one to four hours. The especially preferred protecting groups are groups which can be cleaved by trifluoroacetic acid at room temperature within 1-2 hours.
These protecting groups include such groups as tetrahydropyranyl, xcex2-trimethylsilylethyl, 1-ethoxyethyl, t-butyl, p-methoxybenzyl, 1-adamantyl, diphenylmethyl, triphenylmethyl, trialkylsilyl, (e.g. tri-methylsilyl, triethylsilyl, and the like) trialkylstannyl, (e.g. trimethylstannyl, triethylstannyl, and the like), bis (4-methoxyphenyl)methyl, 2-furanylmethyloxycarbonyl, t-amyl-oxycarbonyl, 1-methylcyclohexyloxycarbonyl, isobornyloxy carbonyl, methoxytrimethylbenzenesulfonyl, pentamethylchromanesulfonyl, 2,4,6-trimethoxybenzyl, 9-xantheneyl and the like.
However, not all of the amino acids have side-chain functional groups. For example, many amino acids have hydrogen, alkyl or aralkyl side chains. These include glycine, alanine, valine leucine, norleucine, phenylalanine, isoleucine and the like. Therefore, these amino acids do not require protecting groups thereon.
To differentiate between those amino acids having protecting groups and those not having protecting groups thereon, the term 
is used. As used herein, if no protecting group is present on the amino acid side chain, such as, e.g., in alanine, (which doesn""t have a functional group and therefore no blocking group is required) xe2x80x9cXxe2x80x9d is xe2x88x92. Moreover, if the amino acid side chain has a functional group, such as in tyrosine, but is unprotected, then this also is indicated by X being defined as xe2x88x92. In other words, in both instances, when X is xe2x88x92, the side group is unprotected.
Although the term functional group is understood by one skilled in the art, it is defined as a group which could react with the reactants used or products formed under peptide forming condition if not protected by a blocking group. These functional groups include amino, carboxy, hydroxy, guanidine, imidazole, amino and the like.
On the other hand, if X is a blocking group, then this signifies that the functional group on the side chain is protected. For example, if AA is serine and X is t-butyl, then the residue 
These protecting groups include the protecting groups described hereinabove.
Abbreviations have been used in the specification and claims with respect to these blocking groups and are listed hereinbelow:
The term amino acid protecting group, as used herein, refers to blocking groups which are known in the art and which have been utilized to block the amino (NH2) group of the amino acid. Blocking groups such as 9-lower alkyl-9-fluorenyloxycarbonyl,2-chloro-1-indanylmethoxycarbonyl (CLIMOC) and benz [f] indene-3-methyloxycarbonyl (BIMOC) and dbd-TMOC are discussed in U.S. Pat. Nos. 3,835,175, 4,508,657, 3,839,396, 4,581,167, 4,394,519, 4,460,501 and 4,108,846, and the contents thereof are incorporated herein by reference as is fully set forth herein. Moreover, other amino protecting groups such as 2-(t-butyl sulfonyl)-2-propenyloxycarbonyl (Bspoc) and benzothiophene sulfone-2-methyloxycarbonyl (Bsmoc) are discussed in U.S. Pat. No. 5,221,754 and the subject matter therein is incorporated herein by reference. Other amino protecting groups are described in an article entitled xe2x80x9cSolid Phase Peptide Synthesisxe2x80x9d by G. Barany and R. B. merrifield in Peptides, Vol. 2, edited by E. Gross and J. Meienhoffer, Academic Press, New York, N.Y., pp. 100-118 (1980), the contents of which are incorporated herein by reference. These N-amino protecting groups include such groups as the FMOC, Bspoc, Bsmoc, t-butyloxycarbonyl (BOC), t-amyloxycarbonyl (Aoc), xcex2-trimethylsilylethyloxycarbonyl (TEOC), adamantyloxycarbonyl (Adoc), 1-methyl-cyclobutyloxycarbonyl (Mcb), 2-(p-biphenylyl)propyl-2-oxycarbonyl (Bpoc), 2-(p-phenylazophenyl)propyl-2-oxycarbonyl (AzOc), 2,2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl (Ddz), 2-phenylpropyl-2-oxycarbonyl (Poc), benzyloxycarbonyl (Cbz), p-toluenesulfonyl aminocarbonyl (Tac) o-nitrophenylsulfenyl (Nps), dithiasuccinoyl (Dts), phthaloyl, piperidino- oxycarbonyl, formyl, trifluoroacetyl and the like.
These protecting groups can be placed into four categories:
1) a base labile Nxcex1-amino acid protecting group such as FMOC, and the like.
2) protecting groups removed by acid, such as Boc, TEOC, Aoc, Adoc, Mcb, Bpoc, Azoc, Ddz, Poc, Cbz, 2-furanmethyloxycarbonyl (Foc), p-methoxybenzyloxycarbonyl (Moz), Nps, and the like.
3) protecting groups removed by hydrogenation such as Dts, Cbz.
4) protecting groups removed by nucleophiles, such as Bspoc, Bsmoc and Nps and the like.
5) protecting groups derived from carboxylic acids, such as formyl, acetyl, trifluoroacetyl and the like, which are removed by acid, base or nucleophiles.
As defined herein, a nucleophile is an electron-rich atom, i.e., an atom which can donate an electron pair, which tends to attack a carbon nucleus but does not act as a Bronsted Lowry base. For example, a nucleophile, as defined herein, includes those molecules which are used for nucleophilic addition across a double bond and behaves in a manner similar to that described in the schemes herein below.
The general mechanism for cleavage of Bspoc and Bsmoc groups are similar in that the nucleophile is believed to react through a Michael-type addition across a double bond. Although the following schemes are shown for Bspoc, it is also illustrative of Bsmoc: 
The nucleophile is believed to attack at the terminal carbon atoms of the propenyl group (Michael acceptor) forming a zwitterion which can eliminate the OCOAA(X)OH anion and H+ to form an alkene-amine and the carbamic acid (8) after protonation. Loss of CO2 will furnish the free amino acid.
The nucleophiles which will function in concert with this invention must have an active hydrogen atom, i.e., a hydrogen atom attached to the nucleophilic atom.
It is preferred that the nucleophile is a simple amine. It is especially preferred that the simple amine is a primary or secondary amine of the formula HNR19R20 wherein R19 and R20 are independently hydrogen, lower alkyl or substituted lower alkyl, the lower alkyl being substituted with OH, CH3, or CH2CH3 or R19 and R20 taken together form a mono or bicyclic ring containing from 4 to 10 ring carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen, sulfur or oxygen.
Typical examples of useful amines include ethanolamine, morpholine, piperidine, diethylamine, 2,6-dimethylpiperidine, piperazine, diethylamine, ethylamine and the like.
An organo mercaptan can also be used as a nucleophile, e.g., alkyl mercaptans, cycloalkyl mercaptans, aryl mercaptan or aralkyl mercaptans. The most preferred mercaptan is benzyl mercaptan. However, when an organomercaptan is used as the nucleophile, the deblocking reaction additionally requires a base catalyst, such as, for example, triethylamine and the like.
The nucleophile can be added as a free compound or as an insoluble reagent attached to a solid support i.e., polystyrene or silica dioxide. These are represented by the formula:
p-[alk]xe2x80x94NuH
wherein p is an organic polymer as defined hereinabove or a silica gel polymer; alk is a chemical bond, alkyl or aroyl chain having from about one to about ten carbon atoms and Nuxe2x80x94H is a nucleophile as defined hereinabove.
A preferred insoluble reagent is the silica based piperazine reagent 9: 
Another useful nucleophile is benzylmercaptan as shown in the following scheme. 
In this scheme the thio-group reacts in a Michael fashion to remove the Bspoc protecting group.
The amino acid fluorides of the present invention can be prepared by art recognized techniques. More specifically, they can be prepared by reacting an N-protected amino acid with the reagent cyanuric fluoride according to the following equation: 
wherein BLK is an amino protecting group as defined herein and X is defined herein. It is preferred that BLK is the FMOC CLIMOC, BIMOC, DBD-TMOC, Bspoc, Bsmoc, or related base sensitive group. This reaction can be run at temperatures as low as 0xc2x0 and up to the refluxing temperature of the solvent, but it is preferred that reaction is run at room temperature. It also can be run in an inert solvent such as pyridine/CH2Cl2 and the like.
The cyanuric fluoride can be prepared from the corresponding chloride in the presence of potassium fluoride at elevated temperatures ranging from 150xc2x0 to 250xc2x0 C., according to the following equation: 
Other fluorinating agents well known in the art, such as thionyl fluoride, 2,4,6-trinitrofluorobenzene, N-methyl-2-fluoropyridinium salts, and the like may be used in place of KF to effect the formation of cyanuric fluoride.
Besides the methods described hereinabove, a new method has been developed to synthesize the protected amino acid fluorides of the instant specification. The new fluorinating agent is a fluoroformamidinium salt and has the formula: 
wherein
R15, R16, R17 and R18 are independently lower alkyl or aryl, aryl lower alkyl, cycloalkyl, cylcoalkyl lower alkyl or R15 and R16 taken together with the nitrogen atom to which they are attached form a 5- or 6-membered ring
or R15, R16, R17 and R18 taken together with the nitrogen atom to which they are attached form a 5- or 6-membered ring or
R15, R16, R17 and R18 taken together with the nitrogen atom to which they are attached form a 5- or 6-membered ring and
A is a counterion.
The aryl, arylalkyl and alkyl groups are as defined hereinabove.
The term xe2x80x9ccycloalkylxe2x80x9d refers to a single ring or a fused ring system containing 3-10 ring carbon atoms and up to a total of 12 carbon atoms. The only ring atom in cycloalkyl are carbon atoms, i.e., there are no hetero ring atoms. The cycloalkyl group may be completely saturated or partially saturated. It may contain one ring or it can contain two, three or more rings. It is preferred that the cycloalkyl group be bicyclic and especially monocyclic. In addition, it is preferred that the ring contain 5-10 ring carbon atoms, especially, 5, 6 or 10 ring carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, decalinyl, indanyl, and the like. The preferred cycloalkyl groups are cyclopentyl and cyclohexyl.
Cycloloweralkyl is an alkylene group, as defined above, bridging the main chain with a cycloalkyl group, as defined herein. Examples include cyclopentylmethyl, cyclohexylmethyl, and the like.
A counterion, as used herein, is an anion used to neutralize the cationic portion of the molecule. Examples include hexafluorophosphate, halide, sulfate, BF4xe2x88x92, sulfite, nitrate, nitrite, acetate, phosphate, oleate, sulide, carboxylate, bisulfate, and the like.
Preferred values of R15, R16, R17 and R18 are lower alkyl and aryl. It is preferred that R15, R16, R17 and R18 are alkyl containing 1-3 carbon atoms, especially methyl or phenyl. It is preferred that at least two and more preferably, three of R15, R16 and R17 are loweralkyl. Examples of the fluoroformamidinium salt of the present invention include tetramethyl fluoroformamidinium hexafluorophosphate (TFFH), trimethylphenylfluoroformamidinium hexafluorophosphate (TPFFH), and the like.
It is also preferred that R17 and R18 and/or R15 and R16 taken together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, e.g., piperidine, pyrrolidine, and the like. It is even more preferred that both R17 and R18 taken together with the nitrogen atom to which they are attached and R15 and R16 taken together with the nitrogen atom to which they are attached form a 5- or 6-membered ring. In both instances, the rings may be unsubstituted or substituted with lower alkyl. It is even more preferred that the ring that is formed between R15, R16 and the nitrogen atom to which they are attached is the same and the ring formed between R18 and R17 and the nitrogen atom to which they are attached are the same. Examples thereof include bis(tetramethylene)fluoroformamidinium, hexafluorophosphate (BTFFH) and the like.
Another preferred embodiment of the present invention is when R15, R16, R17 and R18 taken together form a 5- or 6-membered ring, such as, for example, imidazolidine, hexahydropyrimidine, and the like, which may be unsubstituted or substituted with lower alkyl. An example thereof is 1,3-dimethyl-2-fluoroimidazolium hexafluorophosphate (DFIH), and the like.
The fluoroformamidinium salts of the present invention are prepared in accordance with art recognized techniques. For example, the fluoroformamidinium salts can be prepared from reacting the corresponding chloride salt with a fluorinating agent such as a fluoride, (e.g., alkali fluoride, such as KF, NaF, and the like) or PF631 , and the like in a dry inert polar solvent, such as acetonitrile, and the like. The reaction can be effected at temperatures as low as 0xc2x0 and up to the refluxing temperature of the solvent, but it is preferred that the reaction is run at room temperature.
The chloro derivatives can be prepared in accordance with art recognized techniques, such as the methodology described by Dourtaglou and Gross in Synthesis 572 (1984), the contents of which are incorporated by reference. For example, a urea derivative such as 
is reacted with a chlorinating agent phosgene or oxalyl chloride, and the like, which after CO2 evolution, and addition of the counterion, affords the chloro formadinium salt, wherein R15, R16, R17 and R18 are as defined herein.
The fluoroformamidinium salt of the present invention, as indicated hereinabove, can also be used to synthesize the protected amino acid fluorides of the present invention. More specifically, the protected amino acid fluorides of the present invention can be prepared by reacting an N-protected amino acid with the fluoroformamidinium salt. This reaction is preferably run in an inert solvent, such as chloroform or methylene chloride. This reaction can be run at temperatures as low as 0xc2x0 C. and up to the boiling point of the solvent, but it is preferred that the reaction is run at room temperature. Additionally, it is preferred that the reaction is run in the presence of a base, such as pyridine, triethylamine, and the like.
The amino acid fluorides of the present invention are useful in peptide bond formation. The scope is quite broad, as the amino acid fluorides of the present invention can be coupled with an amino acid, a dipeptide, tripeptide, or higher peptide, having a free terminal amino group. As used herein, the term first amino acid is meant to include amino acids as well as dipeptides, and the higher peptides.
The synthesis of peptides according to the present invention requires the following steps:
1) protection of the carboxyl group on a first amino acid.
2) formation of the amino acid fluorides of the present invention in accordance with the procedure herein.
3) formation of the peptide bond by coupling the amino acid fluoride with the first amino acid.
4) removal of the protecting groups.
A variety of carboxy protecting groups known in the art may be employed. Examples of many of these possible groups may be found in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, by T. W. Green, John Wiley and Sons, 1981, the contents of which is incorporated herein by reference.
The following sequence is illustrative of the coupling of an amino acid fluoride of the present invention with an amino acid having a free amino group: 
In the above scheme, BLK is as defined hereinabove, X1, X2 and X3 are independently defined as x hereinabove, and P is a carboxy protecting group, e.g., methyl ester, t-butylester, xcex2-trimethylsilylethyl ester, benzyl ester and the like.
As shown by the above scheme, the Nxcex1-amino protected amino acid fluoride is reacted with a second amino acid in which the carboxy group is protected. A peptide is formed between the first amino acid and the second amino acid. The peptide chain can be increased by removing the alpha amino protecting group by techniques known to one skilled in the art, and then reacting the corresponding di-peptide with another Nxcex1-amino protected amino acid fluoride to form the corresponding tri-peptide. The Nxcex1-amino protecting group of the tri-peptide is removed and the above cycle is repeated until the desired peptide has been obtained.
The coupling of the N-xcex1 protected amino acid fluoride with the carboxy protected amino acid by the normal two phase technique takes place without racemization.
The present invention can readily be utilized in solid phase peptide synthesis. Solid phase peptide synthesis is based on the stepwise assembly of a peptide chain while it is attached at one end to a solid support or solid phase peptide resin. Two methods are generally well known in the art.
One, the Merrifield method, employs a solid support for attachment of the amino acid or peptide residues. This method employs N-protected amino acids as building blocks which are added to an amino acid or peptide residue attached to the solid support at the acyl (acid) end of the molecule. After the peptide bond has been formed, the protecting group is removed and the cycle repeated. When a peptide having the desired sequence has been synthesized, it is then removed from the support.
The second method, the inverse Merrifield method, employs reagents attached to solid supports in a series of columns. The amino acid or peptide residue is passed through these columns in a series to form the desired amino acid sequence.
These methods are well known in the art as discussed in U.S. Pat. Nos. 4,108,846, 3,839,396, 3,835,175, 4,508,657, 4,623,484, 4,575,541, 4,581,167, 4,394,519 as well as in Advances in Enzymology, 32, 221 (1969) and in PEPTIDES,. VOL, 2, edited by Erhard Gross and Johannes Meienhoffer, Academic Press, New York, N.Y. pp. 3-255 (1980) and are incorporated herein by reference and is fully set forth herein.
During peptide synthesis, it may not be necessary to actually isolate the amino acid fluorides. The protected amino acid fluoride may be prepared in situ and then used in the coupling reaction with the carboxy protected amino acid or peptide. The fluoroformamidinium salts of the present invention permits the skilled artisan to accomplish these goals.
More specifically, in addition, to its use as a source of protected acid fluoride, the fluroformamidinium salts of the present invention can be used as coupling agents for peptide synthesis in which in situ formation of the intermediate acid fluoride precedes the coupling. This approach avoids the need to isolate, purify, and store the acid fluoride, yet allows one to take advantage of the great reactivity of this class of coupling compounds. Furthermore, under these circumstances, there is little, if any, racemization. For example, the coupling reaction of Z-Phe-Val-OH with H-Ala-OMe carried out with the use of tetramethylfluoroformamidinium hexafluorophosphate, in the presence of proton sponge at xe2x88x9230xc2x0 C. gives the expected tripeptide, with only about 1% racemization.
In addition to its use in the syntesis of protected amino acid fluorides and as a simple in situ coupling reagent, the fluoroformamidinium salts of the present invention, such as TFFH, can be used as a coupling agent for assembly of peptides by both solution and solid phase techiques. An example is the synthesis of leucine enkephalin H-Tyr-Gly-Gly-Phe-Leu-OH (SEQ ID NO.: 1) in 53% yield (88.5%) purity) on an automated peptide synthesizer (millipore 9050). Two other examples of solid phase syntheses involved synthesis of the nonamer prothrombin and the 20-mer alamethicin acid, H-Ala-Asn-Lys-Gly-Phe-Leu-Gly-Glu-Val-NH-2 (SEQ ID NO.: 2) and Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Glu-Gln-Phe-OH (SEQ ID NO.: 3), respectively. The latter is unique in that it consists of may hindered amino acids, including xcex1-aminoisobutyric acid (Aib), and could not previously be made by solid phase techniques except via FMOC amino acid fluorides.
The coupling reaction may also contain other additives normally utilized in peptide synthesis such as those utilized to prevent racemization. For example, besides the fluoroformamidinium salts, the following compounds may additionally be added to the coupling reaction: 
and N-oxides thereof and salts thereof wherein
R1 and R2 taken together with the carbon atoms to which they are attached form a heteroaryl ring wherein said heteroaryl ring is an oxygen, sulfur or nitrogen containing heteroaromatic containing from 3 and up to a total of 13 ring carbon atoms, said heteroaryl may be unsubstituted or substituted with lower alkyl or an electron-donating group;
Y is O, NR4, CR4R5;
R5 is independently hydrogen or lower alkyl;
X is CR6R7 or NR6;
R6 or R7 are independently hydrogen or lower alkyl; or R6 and R7 taken together form an oxo group or when n=O, R4 and R6 taken together may form a bond between the nitrogen or carbon atom of Y and the nitrogen or carbon atom of X;
Q is (CR8R9) or (NR8);
when n is 1, R4 and R8 taken together may form a bond between the ring carbon or nitrogen atom of Q and the ring carbon or nitrogen atom of R8;
n is 0, 1 or 2;
R3 is hydrogen;
R8 and R9 are independently hydrogen or lower alkyl or R7 and R8 taken together with the carbon to 95 which they are attached form an aryl ring.
Examples include 1-hydroxy-7-aza-benzotriazole, 1-hydroxy-4-aza-benzotriazole, 1hydroxy-4-methoxy-7-azabenzotriazole 4-N,N-dimethylamino-1-hydroxy-7-aza-benzotriazole, 1-hydroxy-6-azabenzotriazole, 1-hydroxy-5-azabenzotriazole, 1-hydroxy-7-aza-1H-indazole, 1-hydroxyl-7-azabenzo-1H-imidazole, 1-hydroxy-1H-pyrrolo[2,3-b]pyridine, 1-hydroxy-4-t-butyl-7-azabenzotriazole, and the like. In addition, 1-hydroxy benzotriazole could also be utilized as the additive. These compounds as well as other compounds, of Formula II are described in U.S. Pat. No. 5,580,981, the contents of which are incorporated by reference.
The amino acid fluorides of the present invention can exist in various stereoisomeric forms, viz., the D or L stereoisomers. Moreover, the amino acid fluorides may be present in mixture of the D and L forms such as in racemic mixtures. All of these forms are contemplated to be within the scope of the present invention. It is preferred that the stereoisomer of the amino acid fluorides of the present invention exist in the L form.